This PDF file contains the front matter associated with SPIE Proceedings Volume 11223, including the Title Page, Copyright information, Table of Contents, Author and Conference Committee lists.

The evolution of multi-resistant bacteria poses a major threat to our healthcare system worldwide. To address this serious public health issue, the speed and sensitivity of state-of-the-art antibiotic susceptibility testing (AST) such as broth dilution and disc diffusion methods need to be significantly improved to provide better patient care. With the ultimate goal of developing a fast and reliable alternative that outperforms current state-of-the-art procedures, a high-throughput, generally– in terms of bug drug combinations – applicable analytical protocol was developed employing Raman spectroscopy as non-destructive analysis tool to monitor the deuterium uptake of metabolically active bacteria and additionally extract chemical-specific information for bacteria and/or contaminant identification. Our AST is based on two reference strains representing Gram-negative (E. coli) and Gram-positive bacteria (E. faecalis), treated with a total of four different antibiotics. Apart from high sensitivity and specificity, time is the most crucial parameter in clinical diagnosis. Hence, bulk analysis of highly concentrated samples was favored over a single-cell approach to allow timeefficient, straight-forward sample preparation and investigation. The developed AST also comprises a preincubation step (bug-drug incubation prior to the addition of D2O containing medium) which is shown to be key for the development of a reliable test comprising Gram-positive and Gram-negative bacteria. 52 clinical isolates typical for urinary tract infection causing pathogens were investigated in a semi-automated setting showing good agreement with state-of-the-art analytics.

Detecting and characterising bacteria as well as monitoring their viability are routine tasks in microbiology. Conventional methods of cell detection and viability monitoring are often time-consuming and/or expensive. We have developed a near-real time, cost-effective and portable fluorometer, the optrode, for quantifying fluorescence signals. We are currently developing protocols to use the optrode to detect, identify and quantify bacteria as well as to monitor their viability using nucleic acid stains such as SYTO 9 and propidium iodide that are routinely used as live/dead stains. Our results are promising but indicate that better dyes are needed to fully characterise bacteria.

We recently reported the first noninvasive, label free measurement of pH in a bodily fluid in vivo using only Raman spectra i.e. in vivo rat model measurements probing the immediate vicinity of a contusive spinal cord injury (SCI) in the first minutes and hours after injury. Calibrated and assigned using Raman spectra of authentic materials, in the rat model we were not able to sample the cerebrospinal fluid (CSF) to allow comparison with an independent measurement of the pH. Swine presents a better model because they allow physical sampling of CSF, although still not ideal for our purposes. We were only able to physically sample CSF from the fourth cerebral ventricle of 2 different animals, before and after all spectral measurements on cords were completed. One measurement each for 2 different animals on physically sampled CSF averaged a pH of 7.001±0.106 (N=2) as per standard laboratory instrumentation. Using a dynamic analysis and the Henderson-Hasselbalch equation, the average of (N=12) noninvasive Raman-based pH measurements of CSF was 7.073±0.156 and at >95% confidence there is no statistically significant difference between the Raman-based and the physically sampled results. We discuss the difference between the dynamic and static analysis, the implications for our understanding of SCI, the accuracy, precision, calibration, general applicability of this approach and future work.

Several years ago we proposed a new algorithm, PV[O]H, to allow noninvasive in vitro and in vivo spectroscopic analysis of mildly turbid i.e. optically thin or dilute samples that can be considered two phase systems such as plasma and red blood cells (RBCs). The goal was to provide a method for measuring the volume fraction of the sample for each of the two phases noninvasively without physical sampling or labeling. The first application of this approach was to calculate the volume fractions of plasma and RBCs for blood in the peripheral vasculature of skin, to calculate the hematocrit (Hct) of the blood as an early indicator of blood loss. Having validated PV[O]H in completely defined in vitro systems and then for bacterial cultures in various media, we began to envision potential uses for PV[O]H in medicine and biotech applications. Every potential application presents special requirements but nearly all require the embodiment to be physically small and light and have a minimum power footprint. Some applications require retaining the capacity to perform Raman spectroscopy while utilizing PV[O]H and others do not. We describe our first attempt to design and fabricate a small device that can implement PV[O]H without Raman capability. Among many potential uses, we intend PVOH for medical uses involving indications of 1) potential undetected internal bleeding, 2) hematocrit variation during dialysis and 3) infection and inflammation in spinal cord injury. PV[O]H also has potential utility in wellness, exercise science and physiological monitoring of small vessel plasma and red cell volumes continuously over time.

The widespread use of antibiotics has significantly increased the number of resistant bacteria, which has also increased the urgency of rapid bacterial detection and profiling their antibiotic response. Current clinical methods for antibiotic susceptibility testing (AST) rely on culture and require at least 16 to 24 h to conduct. We demonstrate a rapid AST method by monitoring the glucose and/or D2O metabolic activity of live bacteria at the single cell level with hyperspectral stimulated Raman scattering (SRS) imaging.

The current standard for antibiotic susceptibility testing (AST) is based on measuring bacterial growth after 10-24 hours of proliferation. Considering that many life-threatening conditions of infection exist, rapid AST techniques are urgently needed. We developed a rapid AST method based on two-photon fluorescence and coherent anti-Stokes Raman scattering microscopy which can detect antibiotic responses of bacteria within one hour. We used Pseudomonas aeruginosa as a representative pathogen model, and found that antibiotic treatment greatly reduces nicotinamide adenine dinucleotide (phosphate) levels in the bacteria. This enables rapid determination of bacterial susceptibility at the single cell level.

With the rise of antibiotic resistance, phage therapy is seen as a promising alternative to cure infection to multiresistant bacteria strains. However, phage susceptibility tests currently carried out are time-consuming and are not compliant with the automated environment of hospital laboratories. In this work, we present a method for phage susceptibility testing through optical density measurement with the use of lensless imaging technique. Using a 3.3 cm2 area CANON sensor and a custom test card, we are able to simultaneously monitor the bacterial growth or inhibition of multiple bacterial/phage samples and to provide reliable results in less than 4 hour.

Antibiotic resistance is a serious threat to public health. The empiric use of the wrong antibiotic occurs due to urgency in treatment combined with slow, culture-based diagnostic techniques. Inappropriate antibiotic choice can promote the development of antibiotic resistance. We investigated live/dead spectrometry using a fluorimeter (Optrode) as a rapid alternative to culture-based techniques through application of the LIVE/DEAD® BacLightTM Bacterial Viability Kit. Killing was detected by the Optrode in near real-time when E. coli was treated with ampicillin and stained with SYTO 9 and/or PI but only when a suitable concentration, bacterial growth phase, and treatment time was used.

The clinical signs and symptoms of infection in acute and chronic wounds are unreliable. Similarly, swab cultures are inaccurate in this population. Tissue biopsies and polymerase chain reaction (PCR) are more accurate but the results require several days to obtain. As a result, the clinician is forced to treat patients empirically. This has led to the overuse of antibiotics and the failure of advanced therapies due to unrecognized infection. To address this problem a point-ofcare diagnostic was developed to identify bacteria in acute and chronic wounds. The MolecuLight procedure (MiX) exposes the wound bed to violet light at 405 nm. Bacterial fluorophores absorb the light. In turn they fluoresce at specific wavelengths: porphyrins (red) and pyoverdines (cyan). The device detects bacteria in the wound bed at a level greater than 104 by measuring the amounts of red and or cyan fluorescence. A robust body of literature has demonstrated that elevated bacterial levels impede wound healing. The MiX can detect elevated bacteria burden in a wound allowing the clinician to address the infection. In addition, the device can guide advanced wound therapies such as antibiofilm agents, negative wound pressure therapy and preparation of the wound bed for grafting.

Photodynamic inactivation (PDI) is described as a promising therapy for oral inactivation. Due to the low aqueous solubility of curcumin, appropriate delivery systems are required to facilitate its use as a photosensitizer. This study evaluated the effectiveness of on Streptococcus mutans biofilm using curcumin-loaded Pluronic® 127. The micelles were characterized by different techniques. MIC and MBC were determined, and after that the cell viability of the biofilm irradiated by blue LED was obtained by CFU/mL and by confocal microscopy. Curcumin-loaded Pluronic® F-127 micelles can be a viable alternative for curcumin to improve the water solubility and the antimicrobial photodynamic effect.

Photodynamic Therapy (PDT) is an alternative to surfaces decontamination which is based on the interaction between a non-toxic photosensitizer (PS) and a light source suitable for the formation of reactive oxygen species. The objective of this work was to test two new patented devices, the “Photodynamic Inactivation Device” (PID) (MU-BR20.2017.002297- 3) and “Ultrasonic Photodynamic Inactivation Device" (UPID) (MU-BR 20.2018.009356-3), in the photodynamic inactivation (PDI) on contaminated solid surfaces. This device contains low cost light emitting diodes (LEDs) and was built on structures to improve light distribution. This, a low-cost alternative was tested in different microorganisms present on the human microbiota: Staphylococcus aureus, Streptococcus mutans, Escherichia coli and Candida albicans. Results showed that PID caused a significant reduction (p<0.05) of the microbial charge stuck in the orthodontic instruments and the UPID promoted significant reduction (p<0.001) of the microbial in the acrylic plates and titanium disk when compared with the positive control. The new device promoted an effective microbial inhibition on the surfaces tested and, thus, making new studies possible. The perspective is that this new device may be a low-cost and non-toxic alternative to the disinfection of biomedical devices, non-critical instruments and for use in food industry.

Otopathogens such as Moraxella Catarrhalis and Haemophilus influenzae are the major causes of pediatric chronic and recurrent otitis media (OM). This pilot study showed that both M. catarrhalis and H. influenzae were highly susceptible to antimicrobial blue light (aBL) inactivation at 405 nm, either in suspensions and biofilms. Transmission electron microscopy showed aBL-induced damage of cell membrane in M. catarrhalis cells. Ultra-performance liquid chromatography results revealed that protoporphyrin IX and coproporphyrin are the most abundant species of endogenous porphyrins in M. catarrhalis. Our findings suggest that aBL is potentially an effective alternative antimicrobial therapy for OM.

Our aim was to evaluate membrane integrity and the occurrence of possible DNA damage caused by blue light inactivation. We used in our study three bacterial pathogens with distinct biochemical and morphological characteristics: Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa. Through electrophoresis and fluorescence spectroscopy analysis we observed distinct molecular damage between the evaluated microorganisms. Thus, our results suggest distinct mechanisms of inactivation by light blue among different pathogen species. Individual biochemical characteristics of each microorganism may be responsible for the occurrence of distinct structural damage

An important reason for the decreased sensitivity of bacteria towards antibiotics is their capability to form so-called biofilms. The increased tolerance of sessile cells is multifactorial and includes reduced penetration rate and potency of antibiotics through dense biofilms. Strategies that have the ability to interfere with the integrity of biofilms and allowing a better penetration of antimicrobial agents are highly sought after. In this work, we explore the potential of laser-induced vapor nanobubbles (VNB) formed from plasmonic nanoparticles irradiated with nanosecond pulsed laser light to locally disturb biofilm integrity and improve antibiotics diffusion. Our results show that biofilms of both Gram-negative (Pseudomonas aeruginosa) and Gram-positive (Staphylococcus aureus) bacteria can be locally disrupted by the pressure waves from laser-induced VNB inside the biofilms. Most importantly, VNB-mediated biofilm disruption increased tobramycin efficacy up to 1-3 log orders of magnitude, depending on the treatment regimens and type of organism. In addition, we explored the use of VNB to enhance the efficacy of a broad range of antimicrobials used for treating wound infections, towards a first potential clinical application of the technique. Our results confirm that VNB-mediated biofilm disruption is an effective technique to enhance the activity of those antimicrobials that experience hindered diffusion in biofilms. Future work will extend the evaluation of this novel concept towards more complex multi-species biofilms and in vitro wound models before going to in vivo evaluations.

We have developed a technology that facilitates the targeted delivery of glucocorticoids to arthritic joints thereby side-stepping systemic immune suppression while potentially improving efficacy. Our technology loads phototherapeutics inside of red blood cell (RBC) carriers. This ensures that the inactive, RBC-contained phototherapeutic drug is transported throughout the body and released from the RBC carrier only after triggered release by long-wavelength light. We have demonstrated that medications bound to vitamin B12 can be photo-released from RBCs using tissue penetrating, longwavelength red, far-red, and near infrared light in the collagen antibody-induced arthritis model.

Stitches or adhesive patches still represent the gold standard for the closure of wounds. However, inspite of their long history and widespread implementation, these solutions remain problematic in different respects, due to the combination of factors that complicate the healing process, such as foreign body reactions, antimicrobial infections, or the permeability of the repair. Our work consists of the pursuit of an alternative technological platform to seal wounds in different clinical contexts, by the use of laser welding in combination with biocompatible scaffolds made of electrospun fibers containing polysaccharidic components and hosting a variety of dyes, such as FDA-approved indocyanine green or more durable plasmonic nanoparticles. We illustrate the use of these materials in different regimes of optical irradiation, where cw light activates a cascade of photo-thermal and biochemical processes that result into a strong adhesion at the boundary with a connective tissue. We suggest the incorporation of multishell Au@Ag core@shell nanoparticles as a tool serving both as a photothermal transducer and a source of silver cations, which may migrate through the microporous scaffold and exert an antimicrobial function. While the versatility of our materials and methods still leaves substantial room for optimization and even more functionalization, we are confident that our work will make an impact and inspire new synergistic solutions at the crossroads between tissue engineering and biomedical optics.

The rapid evolution of antibiotic resistance increasingly challenges the successful treatment of S. aureus infections. Here, we present an unconventional treatment approach by disassembly its membrane microdomains via pulsed laser photolysis of staphyloxanthin. After staphyloxanthin photolysis, membrane permeabilization, fluidification, and membrane protein detachment, were found the underlying mechanisms to malfunction its defense to several major classes of conventional antibiotics. Through resistance selection study, we found pulsed laser treatment completely depleted staphyloxanthin virulence. More importantly, laser treatment further inhibited development of resistance for several major classes of conventional antibiotics including fluoroquinolones, tetracyclines, aminoglycosides, and oxazolidinones. Collectively, this work highlights a novel platform to revive conventional antibiotics to treat S. aureus infections.

Candida auris, the deadly infectious fungus, was reported to infest nearly 60 hospitals and more than 90 nursing homes in New York City. Moreover, these fungal species have developed resistance to all three major anti-fungal drugs. Drug-resistant Candida spp. and other non-albicans have developed multi-drug resistance around the world. Here, we show that, through efficient photoinactivation of an essential detoxifying enzyme which exists in most of the fungal strains, we could achieve significant eradication of those pathogens by subsequent administration of low-concentration of hydrogen peroxide and antifungal drugs. Noteworthy, hydrogen peroxide or antifungal alone is not effective to eradicate them.

Objectives

This retrospective data-collection study aims to explain how the active matrix metalloproteinase-8-titer (aMMP-titer) influences the immune response of the subject. This is done through monotherapy scaling and root planing (SRP) which is then compared to SRP combined with antimicrobial photothermal therapy (aPTT, Emundo).

Methods

Data collection was monocentric, randomized and split-mouth based. A study group of twenty patients with chronic periodontal disease with a periodontal pocket depth (PPD) 4 mm ≤ PPD ≤ 8 mm, a periodontal screening index (PSI: > 3), and a gingival recession ≤ 2 mm were selected. A diode laser, manufactured by A.R.C. Laser GmbH, with 810 nm wavelength was used. This device implemented three different light transmission systems for transgingival and intra-gingival irradiation. Power settings between 200 and 300 mW were deployed for ten seconds during all treatment steps. The photothermic dye of EmunDo system (A.R.C. Laser GmbH) was infracyaningreen. The adjuvant effect of the antimicrobial photothermal therapy (aPTT) with EmunDo in combination with conventional SRP on the teeth 15 and 35 was compared with the results of monotherapy SRP on teeth 25 and 45.

Results

A reduction of the aMMP-8-titer in gingival crevicular fluid (GCF) was observed in both groups (follow up group and control group) after one month. However; the decrease in the follow up group under SRP in combination with aPTT was significantly more pronounced. The periodontal pocket depths was reduced in both treatment groups. The periodontal probing depth (in mm) shows a larger decrease of the periodontal pocket depth within the follow up group (SPR with aPTT) compared with the control group (SRP).

Conclusion

The aMMP-8-titer showed differences in both groups prior to and after treatment. Active matrix- metalloproteinase-8 (aMMP-8) as a reference parameter for path control in antimicrobial photothermal therapy (aPTT) seems acceptable.

The work is devoted to determining the diffusion coefficient of methylene blue in human gum tissue in vitro using spectroscopic methods. The calculations are based on the application of the free diffusion model, the second Fick law, and the modified Bouguer – Lambert – Beer law. For the first time, the diffusion coefficient of methylene blue into the tissue of the human gingival mucosa in vitro was determined, which, by averaging 10 samples is (1.26±0.34)·10^–7 cm²/s. It was revealed that after the gum is completely stained with dye, the light does not pass into the biological tissue from 200-699 nm, which includes the areas of dye absorption peaks necessary for photoactivation of the dye during photodynamic therapy. The results obtained are important for the personified choice of this method in vivo in the treatment of superficial pathological localizations.